Our immediate goal of this proposal is to investigate the relationship between vascular endothelial growth factor (VEGF) gradient strength and activation of the tip cell phenotype in endothelial cells (the primary cell of the vascular network. Our ultimate goal of understanding this relationship is to maximize in vivo vascular infiltration and anastomosis within a biomaterial implant. The natural extracellular matrix (ECM) contains a heterogeneous composition of biomolecules (not a homogeneous concentration or distribution). This heterogeneity is of particular importance during development of the vascular system in embryos. Engineered ECMs (eECMs) that attempt to recapitulate the natural ECM generally lack this heterogeneity. We believe that this lack is a significant oversight. In particular, we ar excited to focus on instructional chemical gradients to control cell behavior. We believe that the lack of heterogeneity in hydrogels is the reason hydrogel-based in vivo therapies have failed to treat complex maladies, such as chronic and ischemic skin wounds. In this proposal we intend to focus on the activation of the tip cell phenotype, which is the phenotype presented by the leading endothelial cell of an angiogenic sprout. We hypothesize that by activating this phenotype we will subsequently enhance angiogenic sprout infiltration speed and directionality (e.g. into the center of a hydrogel implant). Specifically, in this proposal we will use a hybrid platform of photochemistry and enzymatic chemistry (Hybrid Photopatterned Enzymatic Reaction or HyPER) that we have published to spatially control the covalent immobilization of VEGF within a hyaluronic acid (HA) hydrogel scaffold. In our previous work, we have shown that HyPER allows for both the creation of gradients and immobilization of large growth factors within hydrogels. For this proposal, we will combine these abilities to produce controlled gradients of VEGF. The proposed training plan is a comprehensive study that includes material synthesis, in vitro biochemical and cell behavior analysis, and in vivo studies. We will also include a diabetic mouse colony in our in vivo studies to analyze our approach as a potential therapy. Beyond expanding the knowledge of the trainee and the scientific community-at-large, we believe that this project will provide a route to treating patients with diminished wound healing ability.